During “classical” cell culture in an essentially flat culture vessel, primary cells in general and biopsies in particular tend to de-differentiate. Visibly, biopsies exhibit the ‘melting ice-cream effect’ as cells migrate from a block of tissue out onto the flat supporting surface of the culture vessel. Gene expression is altered in these “migrating” cells, which begin to behave biochemically as isolated cells rather than as cellular components of a differentiated tissue. De-differentiated cells express different biochemical pathways than those normally expressed by corresponding cells in an intact organism. In addition, immortal cells normally have lost some or many of their specialised functions compared to the corresponding mortal cell in the intact organism
In contrast with “classical” cell culture conditions, “microgravity” conditions preserve the differentiation state of many types of cells in culture. Microgravity bioreactors maintain microgravity conditions by continuous rotation of a typically cylindrical or tubular incubation cavity or compartment. This rotation continuously helps to prevent cells from adhering to the walls of the incubation cavity, suspending the cells in a fluid environment using a minimum shear force. This induces them to interact and to aggregate into colonies. These colonies have been given a variety of names including spheroids, cell conglomerates, cell aggregates and ProtoTissue™ (all of which are considered equivalent herein). For microgravity culturing, cells are often initially sown out onto small (ca. 100 μm diameter) beads (this accelerates the formation of microtissue structures) but is not essential and there are several other alternatives published in the literature for example using scaffolds [Lee K W, Wang S, Dadsetan M, Yaszemski M J, Lu L. Enhanced cell ingrowth and proliferation through three dimensional nano composite scaffolds with controlled pore structures. Biomacromolecules. 11:682-9, 2010] or cross-linked hydrogels [Villanueva I, Klement B J, Von Deutsch D, Bryant S J. Cross-linking density alters early metabolic activities in chondrocytes encapsulated in poly(ethylene glycol) hydrogels and cultured in the rotating wall vessel. Biotechnol Bioeng. 102:1242-50, 2009]. As Spheriods are formed by cell growth around these beads, the beads usually become completely covered with cells. Spheriods formed in this manner become highly differentiated so as to resemble adult tissue [Navran S. The application of low shear modelled microgravity to 3-D cell biology and tissue engineering. Biotechnol Ann Rev. 14: 275-296, 2008] [Freed L E, Vunjak-Novakovic G and Langer R. Cultivation of Cell-Polymer Cartilage Implants in Bioreactors. J Cell Biochem. 51: 257-64, 1993][Brown L A, Arterburn L M, Miller A P, Cowger N L, Hartley S M, Andrews A, Silber P M, Li A P. Maintenance of Liver Functions in Rat Hepatocytes Cultured as Spheroids in a Rotating Wall Vessel. In Vitro Cell Dev Biol Anim.; 39: 13-20, 2003].
Microgravity bioreactors have been used in a variety of contexts. Early studies showed that microgravity bioreactor systems helped cells form three dimensional structures by reducing shear stress on the cells [Reduced shear stress: a major component in the ability of mammalian tissues to form three-dimensional assemblies in simulated microgravity. Goodwin T J, Prewett T L, Wolf D A, Spaulding G F. J Cell Biochem. 1993 March; 51(3):301-11].
Now a significant body of literature demonstrates increased differentiation of cells grown in a microgravity bioreactor system. For reviews see: [[Navran S. The application of low shear modelled microgravity to 3-D cell biology and tissue engineering. Biotechnol Ann Rev. 14: 275-296, 2008] and [Growing tissues in microgravity. Unsworth B R, Lelkes P I. Nat Med. 1998 Aug.; 4(8):901-7.] For example, microgravity culturing induces neural precursor cells to form cellular clusters or “neurospheres”. These neurospheres are characterized by a crude, but organized, architecture, having a surface layer of immature proliferating cells (nestin- and proliferating cell nuclear antigen-positive) that enclose strata of more differentiated cells (beta-tubulin III- and glial fibrillary acidic protein-positive). These “neurospheres” show promise for development of neurotransplantable tissue. See e.g. [Neural precursor cells form rudimentary tissue-like structures in a rotating-wall vessel bioreactor. Low H P, Savarese™, Schwartz W J. In vitro Cell Dev Biol Anim. 2001 March; 37(3):141-7.] and see [Rapid differentiation of NT2 cells in Sertoli-NT2 cell tissue constructs grown in the rotating wall bioreactor. Saporta S, Willing A E, Shamekh R, Bickford P, Paredes D, Cameron D F. Brain Res Bull. 2004 December 150; 64(4):347-56.].
Or for another example, microgravity culturing of a multipotential human retinal cell line induced expression of a nearly in vivo phenotype, which could not be achieved when the cells were grown under other conditions [Generation of 3D retina-like structures from a human retinal cell line in a NASA bioreactor. Dutt K, Harris-Hooker S, Ellerson D, Layne D, Kumar R, Hunt R. Cell Transplant. 2003; 12(7):717-31.] Improved differentiation has also been demonstrated in other tissues [Freed L E, Vunjak-Novakovic G and Langer R. Cultivation of Cell-Polymer Cartilage Implants in Bioreactors. J Cell Biochem. 51: 257-64, 1993] [Brown L A, Arterburn L M, Miller A P, Cowger N L, Hartley S M, Andrews A, Silber P M, Li A P. Maintenance of Liver Functions in Rat Hepatocytes Cultured as Spheroids in a Rotating Wall Vessel. In Vitro Cell Dev Biol Anim.; 39: 13-20, 2003]. Some technical problems with microgravity bioreactors have been reported. For example, when temporomandibular joint (TMJ) disc tissues were engineered using either flat culture or a microgravity bioreactor, there were no significant differences in total matrix content and compressive stiffness, notwithstanding marked differences in gross appearance, histological structure, and distribution of collagen types I and II (with the bioreactor discs having more collagen type II). The authors concluded that improvements of the microgravity bioreactor culture system were needed [Detamore M S, Athanasiou K A. Use of a rotating bioreactor toward tissue engineering the temporomandibular joint disc. Tissue Eng. 2005 Jul.-Aug.; 11(7-8):1188-97]. The DNA repair system also seems to be detrimentally influenced [Kumari R, Singh K P, Dumond J W Jr. Simulated microgravity decreases DNA repair capacity and induces DNA damage in human lymphocytes. J. Cell Biochem. 107:723-31, 2009]. Although well known and widely used, currently available microgravity bioreactors have significant limitations:
Another significant limitation of microgravity bioreactors of the prior art is moisture loss, which affects cell growth. Dehydration (even only by 5-10%) during incubation can result in changes in pH and other concentration-dependent parameters, such as concentrations of salts, nutrient substances, and the like. Many cell types are highly sensitive to their environment. For such cells, even a small change in such environmental conditions can influence cell growth and gene expression. This problem is especially pronounced in a small volume bioreactor, where small changes in volume can cause relatively large changes in concentration-dependent parameters. Without some solution to this dehydration problem, a small volume bioreactor would experience rapid loss of moisture, notwithstanding maintenance of humidified conditions (100% relative humidity) in the incubator where the bioreactor was used. This tendency for rapid dehydration in a small volume bioreactor, that is, this tendency for rapid change in relative volume greatly increases the need for time-consuming manual monitoring and manipulation, for example to replenish or exchange culture medium. This tendency effectively renders long-term maintenance of cultures in a small volume bioreactor impractical or impossible. Accordingly, it would be advantageous to provide a microgravity bioreactor with very high relative water retention in the cell incubation compartment.
Still another limitation of microgravity bioreactors of the prior art is that access ports used for adding or removing cells and growth medium have typically relied on conventional “luer lock” closures. These and similar closures have a finite ‘dead’ volume and this becomes proportionally larger as the volume of the bioreactor is reduced. This disadvantage can be circumvented by using ports of essentially no dead volume.
Luer lock closures can also lead to presence of air bubbles in the incubation compartment. Bioreactors are preferably kept free of air bubbles in the incubation compartment which otherwise have detrimental effects, breaking up the Spheriods. This air bubble problem is especially pronounced in a small volume bioreactor, where a single bubble can represent a relatively significant volume. Some solutions to the air bubble problem are known in the prior art. For example, WO 95/07344 provides a reservoir chamber for entrapping gas bubbles away from the incubation compartment. However, these solutions would be wholly unsuitable for a small volume bioreactor because of the volumes involved. A better solution is thus to provide a closure mechanism for access ports that excludes any possibility of introducing air bubbles into the incubation compartment.
Conventional “luer lock” and similar closures also increase fluid turbulence because they do not have a smooth inner surface and this can lead to increased shear forces which will have a detrimental effect on the Spheriods. Microgravity bioreactors require continuous rotation of the incubation compartment to maintain microgravity conditions for differentiated or differentiating cells and other tissues. If the incubation compartment inner surface is not suitably adapted, it may give rise to turbulence. Such turbulence may lead to tearing or “shearing” of Spheriods. Accordingly, it is advantageous to provide microgravity bioreactors with an access port closure mechanism that avoids turbulence. WO 95/07344, U.S. Pat. No. 5,153,131, U.S. Pat. No. 5,437,998, U.S. Pat. No. 5,665,594, U.S. Pat. No. 5,989,913, and U.S. Pat. No. 6,642,019 each disclose improvements of microgravity bioreactors. US 2005/0084965 discloses use of a conventional, commercially available microgravity bioreactor for incubating hepatocyte spheroids. However none of these patents or published applications addresses the problem of dehydration or discloses a microgravity bioreactor having a small incubation compartment volume or having a zero volume access port closure.
Prior art bioreactors of all volumes suffer yet another major problem, namely the difficulty of accessing the incubation chamber/cavity with instruments having dimensions exceeding e.g. a hypodermic syringe or pincers. Thus it is difficult to remove from the bioreactor individual or small amounts of Spheriods without risk of damage (e.g. by shear forces in using a syringe).
U.S. Pat. No. 5,576,211 and U.S. Pat. No. 5,153,131 describe cylindrical bioreactors being rotatable around a central axis, the bioreactors comprising a cell culture chamber and a supply chamber separated by a membrane. U.S. Pat. No. 5,153,131 does not disclose a removable lid, but instead the flange 22 not only constitute the end of the incubator but also the sidewall. Moreover, this flange is by far easy to remove and upon removal it disintegrates from the membrane. U.S. Pat. No. 5,576,211 is not a micro-gravity bioreactor (60 ml incubation volume), but only a roller-bottle system, and hence it will be difficult to access the entire culture chamber, when removing the screw-on ring. Moreover, even when the screw-on ring has been removed there is still a silicone membrane to cope with. Hence the bioreactor system of U.S. Pat. No. 5,576,211 does not provide easy access to the incubation cavity. In FIG. 9 in U.S. Pat. No. 5,576,211, there does not appear to be anything to hold the membrane support grid 82 in medium container 55. Likewise there is nothing to hold the dialysis membrane 64 against 82. Therefore, the user would have to empty or drain 55 before one can open into the cell culture chamber (between the dialysis membrane 64 and a gas exchange membrane in 72). Otherwise the dialysis membrane might fall out and spill the media.
Since microgravity is used to induce the cells to exhibit differentiated phenotypes, the cessation of rotation results in the Spheriods settling usually to the bottom of the incubation chamber. This can induce Spheriods to stick together or to the walls of the incubation chamber and start to lose the desirable phenotype. Thus if it is necessary to remove Spheriods from the incubation chamber, it must be possible to open the incubation chamber, remove one or more pieces of Spheriods and close the incubation chamber quite quickly (e.g. within a few minutes).
Accordingly, it is advantageous to provide an improved microgravity bioreactor that addresses these problems.